Cellular recognition of metal ions and mechanisms of reactivity

PhD ThesisHip replacement is the main therapeutic intervention for end-stage osteoarthritis (OA). Metal-on-metal (MoM) hip implants were introduced to provide a durable alternative to ceramic and polyethylene devices, particularly in a younger patient population. However they are associated with the development of adverse reactions to metal debris (ARMD) which includes osteolysis, soft tissues necrosis, and inflammatory pseudotumours. MoM implants are usually fabricated from a cobalt-chrome alloy. Cobalt activates human Toll-like receptor 4 (TLR4), an innate immune receptor also activated by bacterial lipopolysaccharide (LPS). This study set out to investigate the inflammatory consequences of cobalt-mediated TLR4 activation through a series of in vitro assays developed throughout this work. In human MonoMac 6 macrophages cobalt was found to increase secretion and expression of pro-inflammatory cytokines including IL-8, IL-6 and CCL20. Using IL-8 as a marker of TLR4 activation a small molecule TLR4 antagonist, CLI-095, was shown to inhibit these effects, indicating that they are TLR4-dependent. Similar responses were observed in endothelial cells and osteoblasts. A monoclonal anti-TLR4 neutralising antibody inhibited cobalt-mediated IL-8 expression and secretion, while a polyclonal anti-TLR4 neutralising antibody did not. Further investigation showed that cobalt-mediated TLR4 activation increases expression of intercellular adhesion molecule 1 (ICAM1) and its soluble form sICAM-1. It also promotes primary monocyte and neutrophil migration. A TLR4 mutation did not prevent inflammatory responses to cobalt, although further assay optimisation is required. Co-stimulation of MonoMac 6 cells with cobalt and LPS or nickel caused downregulation of IL6, CCL2 and IL8 expression. Finally, unlike cobalt, chromium and strontium ions did not activate TLR4 and did not induce IL-6 or IL-8 secretion in macrophages. In summary, this study has shown that TLR4 activation by cobalt ions from MoM hip implants results in increased cellular inflammatory responses. The use of TLR4 inhibitors in this study suggests that TLR4 is a potential therapeutic target in ARMD. Overall, the TLR4 signalling pathway is an interesting avenue for further investigation into factors underlying MoM implant failure.Newcastle NIHR Biomedical Research Centre and DePuy Synthes Lt